|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Synonyms:||isoamylase; glycogen 6-alpha-D-glucanohydrolase|
|Concentration:||Supplied at ~ 200 U/mL|
|Expression:||Purified from Pseudomonas sp.|
|Specificity:||Hydrolysis of (1,6)-α-D-glucosidic branch linkages in glycogen, amylopectin and their β-limit dextrins.|
|Specific Activity:||~ 180 U/mg (40oC, pH 4.0 on oyster glycogen) (equivalent to 16 MU Sigma Units/mg)|
|Unit Definition:||One unit of isoamylase activity is the amount of enzyme required to release one µmole of D-glucose reducing sugar equivalent in the presence of oyster glycogen per min at pH 4.0 and 40oC.|
|Application examples:||Applications in carbohydrate research and in the food and feeds, and cereals industry.|
|Method recognition:||AOAC Method 2000.11 and GB Standard 5009.245-2016|
Pure Isoamylase (Glycogen 6-glucanohydrolase) for use in biochemical enzyme assays and in vitro diagnostic analysis. Isoamylase, Fructanase (E-FRMXPD) and Amyloglucosidase (E-AMGDF) are used in the enzyme hydrolysis step of two validated methods for the determination of polydextrose (a low molar mass dietary fiber) in foods: AOAC method 2000.11 and Chinese GB Standard 5009.245-2016.
Please refer to E-ISAMYHP for high purity enzyme suitable for use in starch structural research.
We have a wide range of other CAZyme products available.
Validation of Methods
Salt Tolerance at Vegetative Stage is Partially Associated with Changes in Grain Quality and Starch Physicochemical Properties of Rice Exposed to Salinity Stress at Reproductive Stage.
Sangwongchai, W., Krusong, K. & Thitisaksakul, M. (2021). Journal of the Science of Food and Agriculture, In Press.
Background: Rice yield and grain quality are highly sensitive to soil salinity. Distinct rice genotypes respond to salinity stress differently. To explore the variation in grain yield and grain trait adaptation to moderate, reproductive-stage salinity stress (4 dS/m electrical conductivity), four rice cultivars differing in degrees of vegetative salt tolerance, including Pokkali (salt-tolerant), RD15 (moderately salt-tolerant), KDML105 (moderately salt-susceptible) and IR29 (salt-susceptible), were examined. Results: Grain fertility and 100-grain weight of RD15, KDML105 and IR29, as well as grain morphology of KDML105 and IR29, were significantly disturbed. Interestingly, grain starch accumulation in RD15 and KDML105 was enhanced under stress. However, only RD15 showed changes in starch physicochemical properties, including increased granule diameter, decreased gelatinization peak temperature (Tp) and decreased retrogradation onset temperature (To). Notably, Pokkali maintained productivity, grain quality, and starch properties, while the grain quality of IR29 remained unchanged under salinity stress. Multivariate analysis displayed clear separation of productivity, grain morphology, and starch variables of RD15 in the salt-treated group relative to the control group, suggesting that it was the cultivar most impacted by salt stress despite its moderate salt-tolerance at vegetative stage. Conclusion: Our results demonstrate specific salinity responses among the rice genotypes, and suggest discrepancies between degrees of salt tolerance at vegetative stage versus the ability to maintain both grain quality and starch properties in response to salinity stress imposed at reproductive stage.Hide Abstract
Sequential maltogenic α-amylase and branching enzyme treatment to modify granular corn starch.
Zhong, Y., Herburger, K., Kirkensgaard, J. J. K., Khakimov, B., Hansen, A. R. & Blennow, A. (2021). Food Hydrocolloids, 120, 106904.
Due to the semi-crystalline structure of native starch granules, enzymatic modification of these solid, raw, entities by branching enzyme (BE) is limited. Here, we describe a method to efficiently modify starch by BE after maltogenic α-amylases pre-treatment. This pre-treatment produced pores at the starch granule surface, which decreased the granular yield, but increased the branching degree in starch molecules. BE post-treatments recovered the yield, increased the content of long amylose chains, and the starch crystallinity. WAXS analysis showed that BE transformed the unresolved doublet peak at 2θ 17° and 18° to a strong peak at 2θ 17°, i.e. transformed the granules from the A-type to a mixed A-, B-type allomorph. Syneresis of starch gels increased with increasing BE concentrations and increased the content of slowly digested starch in retrograded starch preparations. Rheology data demonstrated that low and medium BE concentrations produced starch gels with higher G′ and G″ after storage for 1day, whereas high BE concentrations reduced both G′ and G’’. Our data demonstrate the potential of clean, enzyme-based protocols using sequential addition of starch active enzymes for post-harvest modification of raw starch granules to obtain clean and functional starch.Hide Abstract
Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.
Xiang, G., Leemhuis, H. & van der Maarel, M. (2021). Authorea Preprints, In Press.
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two β-strands.Hide Abstract
Influence of microwave treatment on the structure and functionality of pure amylose and amylopectin systems.
Zhong, Y., Tian, Y., Liu, X., Ding, L., Kirkensgaard, J. J. K., Hebelstrup, K., Putaux, J. L. & Blennow, A. (2021). Food Hydrocolloids, 119, 106856.
Pure granular amylose (AM) and pure granular amylopectin (waxy) starch (AP) granules have the high nutritional value in food industry. Effects of microwave treatment (400 W/g DW, 1-8 min) on the structure and properties of transgenic AM granules and AP granules were investigated in direct comparison. Microwave treatment, especially during the first 3 min, decreased the molecular weight of molecules in both the AM and the AP samples. The crystallinity of AM starch initially increased from 15.6% to 20.6%, which was associated with the formation of new Vh-type crystals. After that, crystallinity decreased alongside to 11.3% with the complete disruption of B-type crystals. In contrast, the crystallinity of AP starch initially decreased from 18.9% to 10.8% followed by an increase to 20.0%. Upon prolonged treatment of AM granules, the resistant starch and water solubility was significantly increased. Our data demonstrate notable different microwave-dependent reorganization patterns for pure granular AM and AP molecules as native granular systems, which is helpful to the improvement of functionality of these two starches.Hide Abstract
Supermolecular structures of recrystallized starches with amylopectin side chains modified by amylosucrase to different chain lengths.
Zhang, H., Qian, S., Rao, Z., Chen, Z., Zhong, Q. & Wang, R. (2021). Food Hydrocolloids, 119, 106830.
Understanding the supermolecular structures of recrystallized starches is imperative to manufacture novel starches with controllable physicochemical properties enabling novel applications. In this study, native amylopectin (AP) from maize was modified to different chain lengths using amylosucrase from Neisseria polysaccharea. Native AP granules showed well-organized lamellar structures with a periodicity of ca. 9.0 nm whereas modified starches formed heterogeneous structures with the average crystallite size ranging from 19.77 to 29.88 nm. Acidic treatments eroded amorphous domains in both native AP granules and modified starch particles, producing nanocrystals composed of double helices. A-type nanocrystals (~40 nm) from native AP granules showed quadrangular shape with an acute angle of ca. 60 , while irregular B-type nanocrystals were observed for the modified starches. Molecular characterizations suggested that the nanocrystals had relatively uniform chain lengths (DP 16.5-25.2), and the helix length (the thickness of nanocrystals) was positively correlated to the thermal stability of starches.Hide Abstract
Digestibility of resistant starch type 3 is affected by crystal type, molecular weight and molecular weight distribution.
Klostermann, C. E., Buwalda, P. L., Leemhuis, H., de Vos, P., Schols, H. A. & Bitter, J. H. (2021). Carbohydrate Polymers, 265, 118069.
Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14-76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.Hide Abstract
Dataset on viscosity and starch polymer properties to predict texture through modeling. Buenafe, R. J. Q., Kumanduri, V. & Sreenivasulu, N. (2021). Data in Brief, 36, 107038.
Dataset on viscosity and starch polymer properties to predict texture through modeling. Buenafe, R. J. Q., Kumanduri, V. & Sreenivasulu, N. (2021). Data in Brief, 36, 107038.
Accurate classification tool for screening varieties with superior eating and cooking quality based on its pasting and starch structure properties is in demand to satisfy both consumers’ and farmers’ need. Here we showed the data related to the article entitled “Deploying viscosity and starch polymer properties to predict cooking and eating quality models: a novel breeding tool to predict texture” which provides solution to this problem. The paper compiles all the pasting, starch structure, sensory and routine quality data of the rice sample used in the article into graphical form. It also shows how the data were processed and obtained.Hide Abstract
The role of indica starch in the mechanism of formation of fresh rice noodles.
Yi, C., Zhu, H., Zhang, Y., Wu, S. & Bao, J. (2021). Journal of Cereal Science, 99, 103212.
Starch is the largest component in frequently consumed fresh rice noodles (FRN). The effects of morphology, relative crystallinity, molecular weight distribution and physicochemical (rheological, water hydration and gel texture) properties of starch from five rice cultivars with different amylose contents on the formation of FRN were investigated. FRN made from NR rice cultivar, used for commercial rice noodles making, showed excellent FRN texture. NR starch exhibited larger G′ and smaller tan δ values. The morphology of FRN from GM03 cultivar was closest to NR, but had higher hardness and lower springiness, resilience and cohesiveness. Its starch granules morphology was relatively small and round. The extracted starch from Zhongzao 35, Zao 87 and BX02 rice cultivars, with higher swelling power or lower proportions of amylose (FrI) and higher proportions of amylopectin (FrII), could not produce a dense and consistent FRN structure. It was observed that relative crystallinity of the starch samples had no influence on the formation of FRN. Nevertheless, the gel texture of starch played a role in the formation of FRN. The formation of FRN is the common effect of structure and physicochemical properties of rice starch. The finding of this research would be useful in understanding the role of starch in producing FRN of high quality.Hide Abstract
Influence of physicochemical properties and starch fine structure on the eating quality of hybrid rice with similar apparent amylose content.
Peng, Y., Mao, B., Zhang, C., Shao, Y., Wu, T., Hu, L., Hu, Y., Li, Y., Tang, W., Xiao, W. & Zhao, B. (2021). Food Chemistry, 353, 129461.
In this study, we compared the physicochemical properties and starch structures of hybrid rice varieties with similar apparent amylose content but different taste values. In addition to the apparent amylose content, gel permeation chromatography analysis showed that the higher proportions of amylopectin short chains and relatively lower proportions of amylopectin long chains, which could lead to higher peak viscosity and breakdown value, as well as a softer and stickier texture of cooked rice, were the key factors in determining the eating quality of hybrid rice. High-performance anion-exchange chromatography analyses showed that the proportion of amylopectin short chains (degree of polymerization 6-10) and intermediate chains (degree of polymerization 13-24), which might affect the gelatinisation enthalpy and crystallinity, also contributed greatly to the eating quality of hybrid rice. Moreover, this study indicated that a greater diversity of forms and sizes of starch granules might influence the eating quality of hybrid rice.Hide Abstract
Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae.
Colpaert, M., Kadouche, D., Ducatez, M., Pillonel, T., Kebbi-Beghdadi, C., Cenci, U., Huang, B., Chabi, M., Maes, E., Coddeville, B., Couderc, L., Touzet, H., Bray, F., Tirtiaux, C., Ball, S., Greub, G. & Colleoni, C. (2021). Communications Biology, 4(1), 1-16.
The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.Hide Abstract
Deploying viscosity and starch polymer properties to predict cooking and eating quality models: a novel breeding tool to predict texture.
Buenafe, R. J. Q., Kumanduri, V. & Sreenivasulu, N. (2021). Carbohydrate Polymers, 260, 117766.
Acceptance of new rice genotypes demanded by rice value chain depends on premium value of varieties that match consumer demands of regional preferences. High throughput prediction tools are not available to breeders to classify cooking and eating quality (CEQ) ideotypes and to capture texture of varieties. The pasting properties in combination with starch properties were used to develop two layered models in order to classify the rice varieties into twelve distinct CEQ ideotypes with unique sensory profiles. Classification models developed using random forest method depicted the overall accuracy of 96 %. These CEQ models were found to be robust to predict ideotypes in both Indica and Japonica diversity panels grown under dry and wet seasons and across the years. We conducted random forest modeling using 1.8 million high density SNPs and identified top 1000 SNP features which explained CEQ model classification with the accuracy of 0.81. Furthermore these CEQ models were found to be valuable to predict textural preferences of IRRI breeding lines released during 1960-2013 and mega varieties preferred in South and South East Asia.Hide Abstract
Long chains and crystallinity govern the enzymatic degradability of gelatinized starches from conventional and new sources.
Gaenssle, A. L., Satyawan, C. A., Xiang, G., van der Maarel, M. J. & Jurak, E. (2021). Carbohydrate Polymers, 117801.
Slowly digestible starches have received interest due to their lower increase of postprandial blood glucose and insulin levels and, hence, modification of starches towards slower digestibility has commercial interest. However, chemical characteristics driving enzymatic (digestive) degradation are not fully unraveled. The digestion properties of starches have been linked to their crystalline type, chain length distribution, amylose content or degree of branching, but content and length of relatively long side-chains in amylopectin has not been paid attention to. Therefore, this research focusses on the unique content and length of amylopectin side-chains from conventional and new starch sources (potato, corn, pea, and tulip) correlated to the enzymatic digestion. The rate of hydrolysis was found to be correlated with the crystalline type of starch, as previously suggested, however, the complete hydrolysis of all starches, independent of the crystalline type and source, was shown to be governed by the content of longer amylopectin chains.Hide Abstract
Impact of esterification with malic acid on the structural characteristics and in vitro digestibilities of different starches.
Na, J. H., Jeong, G. A., Park, H. J. & Lee, C. J. (2021). International Journal of Biological Macromolecules, 174, 540-548.
This research focused on the structural characteristics of resistant starches (RSs) that were obtained from corn, potato, and sweet potato and esterified by L-malic acid. Further, the unique effect of the degree of substitution (DS) on the crystalline properties was studied. Different starches were allowed to react with 2 M malic acid (pH 1.5) for 12 h at 130°C. The shapes of the granules and the Maltese-cross shapes of samples were maintained and visible under an optical microscope. The FT-IR spectrum displayed evident carbonyl peaks at 1740 cm−1, and the onset temperature (To) and gelatinization enthalpy (∆H) gradually decreased as DS increased. The malic acid-treated starches exhibited an increased RS content compared to those of the control. The RS contents of potato, sweet potato, and corn, which were 65.5%, 70.0%, and 89.8% in the uncooked MT-samples, decreased to 57.3%, 63.8%, and 86.7% in the cooked MT-samples, respectively, and exhibited high heat stability; corn starch yielded the highest RS among them. The thermal and malic acid treatments resulted in the partial hydrolysis and rearrangement of the helix structure of crystalline area, which was affected by esterification. The result revealed that the RS content increased as that of DS escalated.Hide Abstract
Effects of the degree of milling on starch leaching characteristics and its relation to rice stickiness.
Li, H., Xu, M., Chen, Z., Li, J., Wen, Y., Liu, Y. & Wang, J. (2021). Journal of Cereal Science, 98, 103163.
Rice milling is a very common process to improve rice edible quality. In this study, the effects of the degree of milling (DOM) on starch leaching characteristics and its relation to rice stickiness were investigated. As DOM increased, the whiteness of rice grains and rice stickiness increase, while the protein and lipid content of rice grains decreases. By examining the starch leaching characteristics during rice cooking, it shows that i) increasing DOM significantly increases the total solids and leached starch content while decreases the leached protein content; ii) the molecular size and chain-length distribution (CLD) of leached starch are not significantly varied between rice samples with different DOM, but are significantly different from that of native starch; iii) a significant correlation between rice stickiness and leached amylopectin amount is established; iv) the molecular mechanism for the increased stickiness as affected by DOM is also proposed. The improved understanding of rice processing obtained in this study may allow a better control of rice eating quality.Hide Abstract
Interaction with longan seed polyphenols affects the structure and digestion properties of maize starch.
He, T., Wang, K., Zhao, L., Chen, Y., Zhou, W., Liu, F. & Hu, Z. (2021). Carbohydrate Polymers, 256, 117537.
This study investigated effects of longan seed polyphenols (LSPs) on the structure and digestion properties of starch, and discussed the interaction mechanism between starch and LSPs. The results showed cooking with 20 % LSPs did not change amylopectin chain length distribution of normal maize starch, however, the amylose content was reduced from 21.60 to 14.03 %. This suggests LSPs may interact with starch via non-covalent bond. Isothermal titration microcalorimetry and XRD results confirmed the existence of non-covalent interaction, and indicated that LSPs may enter the hydrophobic cavity of amylose, forming V-type inclusion complex. LSPs did not affect gelatinization temperatures of maize starch, whereas 20 % LSPs decreased the enthalpy change by about 26 %. The digestion results indicate significant inhibition effect of LSPs on the digestion of cooked starch, attributing to the interaction of LSPs with starch. These suggest potential applications of LSPs as functional ingredients in modulating postprandial glycemic response of starchy food.Hide Abstract
Causal relations among starch fine molecular structure, lamellar/crystalline structure and in vitro digestion kinetics of native rice starch.
Li, C., Hu, Y., Gu, F. & Gong, B. (2021). Food & Function, In Press.
Native rice starch is a source of slowly digestible starch in e.g. low-moisture baked products, while the molecular and lamellar/crystalline structure giving rise to this low-digestibility property is still largely unknown. In this study, the in vitro digestion kinetics of 11 rice starches with a wide range of amylose content were investigated. Applying the logarithm of slope (LOS) plot to the starch digestograms suggested that only a single first-order kinetics phase existed. More importantly, results for the first time showed that rice starches with shorter amylopectin short chains (DP 10-26) had more perfectly aligned crystalline lamellae and much slower digestion rates than the other starches. Interestingly, no correlations were found between the starch lamellar thicknesses with its digestion rate. It suggests that lamellar perfection plays a dominant role in the determination of native starch digestibility. Furthermore, starches with relatively shorter amylose short and medium chains showed a significantly higher amount of V-type amylose-lipid complex, and smaller maximum digestion extent. These results could help the rice industry develop a new generation of rice products with slower starch digestion rate and more desirable nutritional values.Hide Abstract
Impact of amylose content on the starch branch chain elongation catalyzed by amylosucrase from Neisseria polysaccharea.
Wang, R., Li, Z., Zhang, T., Zhang, H., Zhou, X., Wang, T., Feng, W. & Yu, P. (2021). Food Hydrocolloids, 111, 106395.
In this study, three rice starches with different amylose content were isolated from three rice varieties, followed by chain elongation using amylosucrase from Neisseria polysaccharea (NpAS). Herein, the chain elongation could induce the starch precipitation during the enzymatic reaction. With a higher content of amylose, an earlier precipitation occurred, leading to the decrease of transglycosylation efficiency of the enzyme. Results from X-ray diffraction and thermal property analysis indicated that the crystalline structure of the NpAS-modified starches largely formed at the precipitation point. The elongation at the surface of the starch precipitates, however, would not enable the formation of crystallites. In addition, the amylose might participate in the recrystallization of starch molecules. Digestion kinetics revealed that all of the NpAS-modified starches contained rapidly and slowly digested fractions, the latter of which had a digestion rate being dependent on the structural stability of crystallites (length of double helices). These findings may provide an efficient way to produce chain-elongated starches with desirable functionality and digestibility.Hide Abstract
The molecular structure of starch from different Musa genotypes: Higher branching density of amylose chains seems to promote enzyme-resistant structures.
Yee, J., Roman, L., Pico, J., Aguirre-Cruz, A., Bello-Perez, L. A., Bertoft, E. & Martinez, M. M. (2021). Food Hydrocolloids, 112, 106351.
Starch from bananas/plantains, belonging to the genus Musa spp, is gaining prominence given its great potential as a healthy food ingredient made from an inexpensive raw material. Recent works highlight the outstanding potential of Musa starch to develop enzyme-resistant structures upon retrogradation. However, despite the wide variety of Musa cultivars (due to both natural mutation and breeding selection), there is no comparative investigation of the starch molecular structure from the most commonly cultivated Musa genotypes. In this work, the starch from six Musa cultivars harvested during the same growing season from the same parcel, was purified and analyzed for amylose ratio, amylose chain length distribution, and amylopectin unit and internal chain length distribution. Results showed significant differences between the fine structure of all Musa amylopectin molecules, which were structurally categorized as type 4 (consisting of a high number of B3-chains, few BS- and Bfp-chains, and low S:L and BS:BL ratios). Moreover, the different Musa starches exhibited dramatic differences in amylose ratio (17.7-27.6%), amylose branching degree (as evidenced by differences in the population of short chains of approximately 260 glucose units, GU) and a shorter average length (approximately 1000 GU) of the population of long amylose unit chains. Remarkably, these differences in amylose structure resulted in the cultivar Manzano (Musa AAB, silk subgroup) to possess a dramatically lower extension of in vitro starch digestion (C90 = 4.70%) than the rest of the cultivars (C90 = 17-18%) after full gelatinization and retrogradation for 7 days.Hide Abstract
Characterization of resistant waxy maize dextrins prepared by simultaneous debranching and crystallization.
Lee, D. J., Kim, J. M. & Lim, S. T. (2021). Food Hydrocolloids, 112, 106315.
Waxy maize starch was debranched using isoamylase and simultaneously crystallized in the same batch at 50°C for 7 days. The crystallized starch dextrins were isolated as precipitates and characterized for chain length distribution, crystallinity, thermal transition property, morphology, and in vitro digestibility. The long B chains released from the amylopectin crystallized more readily than the short A chains. The dextrins exhibited a typical A-type crystalline arrangement with high thermal stability. The recovery yield from the waxy maize starch was 70.4% after 7 days. The amount of resistant starch (RS) in the crystallized dextrin treated for 7 days was 83% (uncooked) or 46% (cooked). The simultaneous debranching and crystallization process was effective for the formation of dextrin crystals from waxy maize starch with thermal stability and good resistance to digestive enzymes.Hide Abstract
Generation of short-chained granular corn starch by maltogenic α-amylase and transglucosidase treatment.
Zhong, Y., Keeratiburana, T., Kirkensgaard, J. J. K., Khakimov, B., Blennow, A. & Hansen, A. R. (2021). Carbohydrate Polymers, 251, 117056.
We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.Hide Abstract